The goal of the PPG (Gene Therapy for Cystic Fibrosis) is to create gene transfer vectors that will efficiently transduce cells of the lung. Major but not exclusive therapeutic targets are the epithelia of the large and small airways, which are sites of cystic fibrosis lung disease. The major hypotheses tested in the PPG are: (1) new vectors are needed, including higher capacity, better expressing AAV vectors; high titer, safe lentiviral vectors; and adenoviral vectors specifically targeted to airway epithelial receptors; and (2) that a rate-limiting variable for gene transfer efficiency in the lung is at the site of initial vector-cell interaction, including both binding and entry across the plasma membrane. Three Projects and four Cores are proposed. Project I (Parvovirus) Vectors for Airway Delivery, R.J. Samulski, P.I.) proposes to design and produces new AAV vectors that increase the vector packaging size, augment the efficiency of vector entry, and increase the efficiency of expression (conversion from single strand to double strand DNA templates) using chimeric virion capsids, targeting ligands and modified viral terminal repeats. (Equine Lentiviral Vectors for Gene Delivery, J.C. Olsen, P.I.) proposes to develop high titer, efficiently expressing, and are equine lentiviral vectors. The important targets for the lentiviral vectors will be airway epithelial cells, which throughout the airways exhibit low rates of proliferation. Project III (Cell Biology of Airway Epithelial Gene Transfer, R.C. Boucher, P.I.) proposes to define the barriers and targets in the apical domain of airway epithelia, modify the barriers using either oxidant injury or more specific modulators of the tight junctions, and finally, modify vectors to target a class of receptors on the apical membrane that exhibit cellular internalization in response to agonist addition. The Projects are supported by the Administrative Core; the Cell Culture Core that will supply human airway cultures to all Projects, small airway cultures, and smooth muscle cells, the Vector Core that will generate AAV, lentiviral and adenoviral vectors (radio-labeled/fluorescently labeled); and the Morphology Core that will provide histology, electron microscopy, and confocal microscopy. The PPG is a highly interactive program designed to modify vectors and test their interactions with target cells in vitro and in murine models in vivo. Achievement of the PPG goal will be to bring to the next clinical trials vectors that are safer and more efficient for the treatment of lung disease.